1 Report information

1.1 Data set information

Data set: 2021-231_FASP_filters

User(s):

Date: 2023-07-19

Notes:

Objective: Evaluate the risk of possible contamination with polymers and small molecules in the samples from the data set.

1.2 HowDirty evaluation of LC-MS results

This report was generated using HowDirty. This is meant to help you evaluating How Dirty are your samples (or system) in LC-MS analyses. The possible contaminants reported hereby could impact the quality of the results (e.g. contaminant IDs and reproducibility) and, in extreme cases, even damage the column or instruments. The raw data are previously analyzed using Skyline to extract LC-MS peaks possibly corresponding to contaminants. Then, a Risk level is assigned to each contaminant by comparing against the reference threshold (see Procedures for details). Finally, the Risk is similarly assessed at the Sample and whole Sample Set levels. These are indicative of the level of possible contamination.

Code author: David Gomez-Zepeda (HI-TRON)

Version: 0.04

2 Procedure

2.1 Data analysis

  • Raw data was imported into Skyline to extract the features corresponding to possible contaminants (ToDo: add references)
  • For this purpose, the Molecular Contaminant List template was used (Gomez-Zepeda et al., 2023; modified from Rardin, 2018)
  • The feature area of extracted ion chromatograms was processed using HowDirty to generate this report (Gomez-Zepeda et al., 2023)

2.2 Warning

The algorithm used for peak picking in Skyline is simple and based only on m/z and charge (z). Thus, some true contaminant peaks (e.g. peptides) could be incorrectly assigned to contaminants (i.e. false positives). Therefore, it is recommended to also look into the Skyline file to evaluate other factors, such as patterns of contaminant groups elution across the retention time.

2.3 Calculations within HowDirty

  • TICA = Total Ion Current Area
  • Abundance (Normalized abundance) = TotalAreaMS1 / TICA
  • TotalAbundance_ContaminantGroup = Sum (Abundance_ContaminantGroup) across all the contaminants in one ContaminantGroup for one sample
  • Abundance_total = Sum (Abundance) across all the contaminants for one sample
  • Contaminant-specific Risk level assessment was performed by comparing the Abundance of the possible contaminants in each one of the test samples (current data set) against thresholds previously extrapolated from a reference data set (~ 1000s runs). These thresholds are reported in results/2021-231_FASP_filters_report_contaminants_20230719_1911.xlsx–> ref_conta_tshd
  • Sample level summary contaminant group assessment was performed by comparing the TotalAbundance_ContaminantGroup against the summed thresholds from each contaminant
  • Sample level summary contaminant risk assessment was performed by comparing the Abundance_total against the sample-level quantile thresholds from the reference dataset
  • These thresholds are reported in results/2021-231_FASP_filters_report_contaminants_20230719_1911.xlsx–> ref_conta_tshd_sample

3 Input

The data set contained the following:

Variable Count
Conditions 3
Samples 45
ReplicateNames 45
ContaminantGroups 17

4 Summary risk evaluation (total contamination)

4.1 Global

  • The piechart below shows the percentages of samples associated to each risk level
  • The results were exported to results/2021-231_FASP_filters_report_contaminants_20230719_1911.xlsx –> risk_summ_sampleset
  • RiskLevel = “1) Very Low”, “2) Low”, “3) Medium”, “4) High”, “5) Very High”, “6) No threshold in reference”

4.2 Conditions boxplot

  • Statistical difference was assessed by a Wilcoxon signed-rank test

4.3 Sample (ordered by name)

  • The table can also be found in results/2021-231_FASP_filters_report_contaminants_20230719_1911.xlsx –> conta_summ_sample.

4.4 Sample (ordered by abundance)

  • The table can also be found in results/2021-231_FASP_filters_report_contaminants_20230719_1911.xlsx –> conta_summ_sample.

5 Contaminant group

5.1 Condition dotplot

5.2 Sample dotplot

5.3 Sample boxplot

6 Contaminants per sample

6.1 Abundance boxplot

6.1.1 PEG

6.1.2 Phthalate esters

6.1.3 Polysiloxane

6.1.4 Others

6.1.5 Triton X-100

6.1.6 PPG

6.1.7 Tween 20

6.1.8 Triton 101

6.1.9 DECYL-PEG

6.1.10 n-Nonyl PEG

6.2 Pseudochromatograms (Abundance vs. RT)

6.2.1 PEG

6.2.2 Phthalate esters

6.2.3 Polysiloxane

6.2.4 Others

6.2.5 Triton X-100

6.2.6 PPG

6.2.7 Tween 20

6.2.8 Triton 101

6.2.9 DECYL-PEG

6.2.10 n-Nonyl PEG

6.3 Table

  • The following table shows the results of possible contaminants detected in each sample.
  • An extended version of this table containing the Area and Total-Ion-Count-Area (TICA) can also be found in results/2021-231_FASP_filters_report_contaminants_20230719_1911.xlsx –> conta.
  • RiskLevel = “1) Very Low”, “2) Low”, “3) Medium”, “4) High”, “5) Very High”, “6) No threshold in reference”

7 Appendix

8 References